Hereditary angioedema develops in individuals heterozygous for C1 inhibitor (C1INH) deficiency. The product of a single allele is insufficient to control activation of the proteolytic systems normally regulated by C1INH. A logical approach to therapy is to enhance inhibitor synthesis from this single gene. This proposal will examine three aspects of C1INH synthesis regulation that have not been studied extensively in any gene and that remain poorly understood. Specific Aim 1 will examine the role of the polypurine-polypyrimidine (Pu-Py) segment of the C1INH promoter. We will test the hypothesis that enhanced transcription mediated by this region results from interaction of transcription factors with specific sequences within the Pu-Py segment rather than via H-DNA (triple helix hinged) formation. Preliminary electrophoretic mobility shift assays and supershift experiments have demonstrated interaction of HNF-1alpha (hepatocyte nuclear factor) with one site in addition to several other, as yet unidentified, nuclear proteins that bind to sites within the region containing the Pu-Py sequence. Further studies will identify and characterize these proteins and their function. Other studies suggest cooperativity between the Pu-Py region and the HNF-1alpha site. We will test the hypothesis that this cooperativity results from interaction between transcription factors that bind to these regions by co-immunoprecipitation and by direct isolation. DNAse hypersensitivity experiments will be performed to provide support for the hypothesis that H-DNA formation takes place in vivo, as will experiments to analyze induction of transcription using mutated promoter constructs that are incapable of H-DNA formation. Lastly, nucleosomal reconstitution experiments will test the hypothesis that triplex formation creates a nucleosomal barrier during replication. Specific Aim 2 will examine the role of phosphatases in down-regulating interferon (IFN)-gamma-mediated induction of C1INH in hepatocytes in comparison with the role of proteosome degradation or binding of STAT1 by specific inhibitory proteins. Preliminary data suggest that phosphatases play a major role in this down-regulation. This hypothesis is unexamined in hepatocytes, which are the primary source of most plasma proteins, many of which are IFN-responsive. Specific Aim 3 will test the hypothesis that estrogens suppress C1INH transcription. Furthermore, we hypothesize that the therapeutic effect of androgens is a result of down-regulation of estrogen receptor expression, which reverses the estrogenic effect. The studies proposed here will contribute both to knowledge of the regulation of C1INH itself and to the role of hormones, nuclear phosphatases and Pu-Py sequences on gene regulation in general.